20.109(F10): Mod 2 Day 3 Tools for system engineering

From OpenWetWare

Revision as of 13:15, 18 October 2010 by Nkuldell (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search
20.109(F10): Laboratory Fundamentals of Biological Engineering

Home        People        Schedule Fall 2010        Assignments        Lab Basics        OWW Basics       
DNA Engineering        System Engineering        Biomaterials Engineering              

Contents

Tools for system engineering

Introduction

Biological systems are not static. Thus they can be engineered to account for changing environmental conditions, as we've seen through our examination of two component regulatory systems. In addition, they can be engineered to account for changes that occur as the cells replicate and divide over time. Indeed, evolution of biological systems away from an original specification can be viewed as a curse (it's not like computer scientists have to worry that their software programs change functions when they relaunch them!) or a blessing (evolution can find a solution we didn't ever dream of). Here we'll take the rosy view and try to harness genetic variability to improve the bacterial photography system. In particular we'll screen a library of changes in the Cph8 gene to find ones that increase the kinasing activity of the sensor when the cells are growing in the dark. The mutants (we'll call them K+) should make the "dark" color of the photographs more dark by increasing the amount of phosphorylated OmpR that stimulates LacZ transcription.

The region of the Cph8 protein to focus on for this purpose has been defined through traditional scientific studies of EnvZ, for example the work from Tom Silhavy's lab( PMID: 9721293 and pdf here). We've also been guided by the expertise of MIT's Mike Laub, whose lab studies the specificity and rewiring of two component regulatory systems. From these sources, a span of 5 contiguous amino acids can be identified as relevant for shifting the balance of EnvZ to greater kinasing or greater phosphatasing activity. These five residues in EnvZ are Alanine at amino acid 239 ("A239") through Histidine at amino acid 243 ("H243"), where mutations in the flanking residues (A239 and H243) have been shown to enhance the phosphatase activity of EnvZ and mutations in the internal residues (G240 V241 S242) enhance the kinase activity of EnvZ. The amino acid changes that modify the enzymatic activities are indicated on the figure below. Two important notes about these mutations though: First, the balance of kinase to phosphatase activities have been affected by the changes, but the mutations do not shift the reactions to fully "on" or fully "off." Second, the fusion protein of Cph1 to EnvZ, called Cph8, changes the numbering of the residues, as shown in the figure below. It's hoped, however, that the local environment of the region is similar to the natural EnvZ protein. Image:EnvZ,Cph8 align.png
To complement the genetic approach for solving biological engineering puzzles, we'll also consider three other approaches in synthetic biology. The first is a Registry of Standard Biological Parts, essentially a community resource that has some ready-made and useful genetic elements that can be assembled into synthetic biological devices systems. The second approach is to use the Tinkercell computer model to simulate the behavior of the bacterial photography system, with the goal of performing "in silico" experiments that would take days or weeks to do at the bench. And finally, in the third approach, we'll recapitulate the genetic network of the biological system using electronic components, making explicit some of the benefits and limitations of such an approach and the often-cited analogy between building with biology and building with computer programs or electronic components.

Protocols

Before you leave today, you should examine the bacterial photograph you set up last time and document your work and your ideas about the experiment.

Part 1: Library Screen

The details for how the libraries were constructed and what kinds of changes are reasonable to expect will be considered in the next lab session. For today, you will transform a pool of DNA with degeneracies in the positions that affect the kinasing activity of EnvZ. The recipient bacterial strain is identical to the bacterial photography system except that it does not harbor a plasmid encoding the light-sensing fusion protein Cph8. It does encode the OmpR-regulated LacZ gene as well as the phycobilins from a plasmid.

To carry out the transformation you will try your hand at a different technique for getting DNA into cells, namely electroporation. This method involves exposing a small but dense volume of cells to a pulse of current. The pulse momentarily flips the lipid bilayer, opening small spaces for the DNA to pass into the cells. As you can imagine, the cells aren't fond of such treatment and the single most important step to help them recover is to quickly add media to the cells once they've been electroporated. The process is also very sensitive to salts in the DNA, and if you pipet too much DNA on to the cells, the extra salt may cause an electrical "arc," (you'll know this has happened from the flash of light and the "pop" you'll hear) frying the cells dead. If this happens, please get another aliquot of cells from the faculty and try the electroporation again, with less DNA.
  1. When you are ready to electroporate the library, retrieve an aliquot of cells from the teaching faculty, a sterile cuvette, and an aliquot of rich, pre-warmed "SOC" media.
  2. Put the cuvette on ice.
  3. Pipet 2 ul of the library DNA that is being held in an icebucket on the teacher's bench into your aliquot of cells.
  4. Let the cells and the DNA incubate on ice one minute.
  5. Transfer 50 ul of the cells (or more if the tube has more volume) to the chilled cuvette and recover it with the blue lid.
  6. Put on your safety goggles.
  7. Tap the cuvette on the bench so the cells rest in the bottom of the cuvette.
  8. With the cuvette's "nub" facing away from you, slide the cuvette into the electroporation chamber. Push the slide into the chamber until the cuvette is between the metal contacts. The lid on the cuvette will seem to block the path but in fact, it doesn't block the slider if you've lined thing up.
  9. Make sure the electroporator is set to "Ec2"
  10. Hold the pulse button until you hear a beep. Listen carefully since the beep is not loud.
  11. Quickly remove the cuvette from the holder and immediately add the 0.5 ml volume of "SOC" media to the cells. Delaying this addition by even 1 minute has been seen to decrease transformation by 3 fold.
  12. Transfer the cells and the media back to an eppendorf tube and place the tubes on the nutator in the 37° incubator for 1 hour. During this incubation you can work on Parts 2, 3 and 4 of today's protocols.
  13. Spread 50 ul of the electroporation mix onto a Tetrazolium+Cam34+Amp200 petri dishes. Plate 300 ul of the electroporation mix on another Tetrazolium+Cam34+Amp200 petri dish. One of these two dilutions should have single, well-isolated colonies to examine next time. Incubate the plates at 37° in the dark until next time.

Part 2: Registry of Standard Biological Parts

What would it take to make DNA serve as a low-level programming language so that a genome is simply a particular program?

  • DNA, like software, has an alphabet but with only four letters in the genetic code.
  • Since there are proof-reading mechanisms in the "hardware," i.e. in the cell, syntax errors may be less likely to arise than in Python or Perl or C++.
  • The code for cellular programs is messy but, honestly, so are computer programs. Subroutines are often dependent on one another (the cell cycle and DNA replication for example) and parts of the program get reused in useful, but complicated and unpredictable ways (seen as cross-talk in signaling pathways for example).
  • Genetic code and computer code are both susceptible to viruses that highjack normally benign functions.

The analogy of the DNA as computer code is not perfect. We have to set aside the presumption of an intelligent agent responsible for writing the initial program as well as accept that natural events will change the code over time (evolution leading to genetic variation--the very thing we're trying to harness in the first part of today's lab). And no good tools exist for systematically debugging the genetic code.

What would make genetic code easier to write? One idea is to make it a more “object oriented” language, defining units of known function that could be combined in standard and predictable ways. One effort to facilitate genetic programming can be found at The Registry for Standard Biological Parts. The Registry is a catalog of parts that describe basic biological functions. For example BBa_B0010 is the part number for a transcriptional terminator. The Registry of Standard Biological Parts makes its parts freely available to interested researchers and engineers, and allows registered users to add and annotate parts.

To familiarize yourself with the Registry, you'll design a protein-generating device for E. coli. This device will consist of at least 3 parts from the Registry: an ORF, a promoter and a ribosome binding site (RBS).
Registry Search Features
Registry Search Features
Finding a protein coding part

Try using the "catalog of parts and devices" (the leftmost icon here) to search for a part you'd like to use. Start your search in the "protein coding sequences" and then identify one of interest to you and your lab partner.

  • What part number have you chosen?
  • Follow the link to the data page associated with part. Is anything specified about its use or performance?
Finding a promoter part

Try finding this part with the "search parts" function (under the Registry tools list on the right-hand side of the page that's here.)

  • Did you search by text, by part number, or by subpart? What worked and how well?
  • What part number have you chosen?
  • What can you learn about the regulation of transcription from this part's number? part's name? part's information at the Registry?
  • Follow the link to the data page associated with this part. Is anything specified about its use or performance?
Finding an RBS

To find an RBS you should restrict your search to a set of the Registry's well-characterized ones from Chris Anderson's lab or the community collection, several of which are from Ron Weiss' lab.

  • What part number did you choose?
  • Can you anticipate the strength of this RBS in your three part construct? Why or why not?

NOW: In plain English, what behavior do you expect for your three part construct? You should include this answer in your lab notebook and if you choose to do the optional "for next time" assignment, you can use this example as a point of departure for your discussion.

Assembly of this construct

Part of the usefulness of the Registry is that the parts conform (mostly) to a standardized assembly scheme. This scheme enables the same restriction enzymes to be used to physically piece together parts in series. The scheme does not ensure functional assemblies, however. If you have time before your cells are ready today or before the electronics exercise, then you can familiarize yourself with this "BioBrick™ assembly scheme."

Part 3: CAD tool for synthetic biology

Revisit the visual model for the bacterial photography system that you assembled using Tinkercell last time. Today you will run a deterministic simulation according to slides 16-23 in the linked tutorials found here and here.

Part 4: Modeling biological versus electrical devices

In this section, we'll explore the issue of gain and signal strength in the context of an electrical circuit. As you work through this exercise, consider how the lessons learned from experimenting with an electronic circuit would map to the engineering of biological systems.

starting kit
starting kit

Acknowledgements

Many thanks to Tom Knight, Reshma Shetty and Barry Canton who motivated and designed an early version of this exercise. And a special shout out to Steve Wasserman for his guidance in redeveloping, troubleshooting and building this circuit to more directly reflect the experiment we're performing.

Safety information

In this exercise, you'll be working with circuits connected to a battery. Practice good habits by never touching the circuit without first unplugging it.

    • also please note:

When you connect the battery to the breadboard, make sure you do so in the correct orientation for the circuit (red to red, black to ground) or the OpAmp will break...really.

System design

A diagram of the electrical circuit that is analogous to the bacterial photography system. Circuit designed by Steve Wasserman
A diagram of the electrical circuit that is analogous to the bacterial photography system. Circuit designed by Steve Wasserman


This system is fairly simple one since it only consists of a few components. In contrast to the bacterial photography system in which the signal is propagated through protein activities, here signals are propagated as either voltage or current. As you can see in the schematic, the circuit contains the following parts.

  1. Photodiode: #LT959X-91-0125: a light sensor (mimics Cph8 and the phycobilins). When light shines on the photodiode, its resistance is decreased and current flows through it.
  2. OpAmp: #AD8031ANZ: a logic device that detects a difference in + and - current inputs and amplifies it.
  3. Resistor: components which resists current flow by producing a voltage drop across it. The gain in this system is proportional to the resistance, and by varying the resistance into the OpAmp, the sensitivity of the circuit to light can be tuned.
  4. LED: a device with a detectable output (mimics LacZ). A voltage drop across its terminals turns the green-colored light on. The 820Ω resistor is added to the circuit before the LED to ensure that the voltage drop across the LED isn't too high (which can cause the LED to breakdown).
Photodiode (sensor) OpAmp (logic) Resistor (gain) LED (actuator)

Let's start building

breadboard connections OpAmp wiring diagram finished circuit

The circuit has been constructed using a breadboard which is a convenient way to construct electrical circuits. The breadboard holes are connected beneath the plastic as shown in the photo. Take note of these connections because they'll affect how you will connect up components in this exercise.

  1. Power the breadboard by running a wire from the +V source (= the red terminal) to the + rail (= the red + connections at the "top" of the breadboard).
  2. Run the - rail (= the blue - at the "bottom" of the breadboard) to the black, ground terminal.
  3. Use a small wire to connect the A-E rails to +9V.
  4. Use a small wire to connect the F-J rails to ground.
  5. Position the OpAmp across the trench in the breadboard.
  6. Power the OpAmp by connecting it to the +9V and ground using the pin diagram that's in the spec sheet and is reproduced here. Note that the small dot on the corner of the OpAmp indicates pin #1.
  7. Connect the OpAmp's + input (pin 3) to ground.
  8. Connect the OpAmp's - input (pin 2) to the photodiode. NOTE: the photodiode is asymetric and must be inserted into the breadboard so that the leg under the "flat" edge is to ground. Leave some space between the photodiode input and the OpAmp so an additional resistor can be added to the circuit later.
  9. You'll make 2 connections to the OpAmp's output (pin 6)
    • The first connection from the OpAmp's output should be to the 820 ohm resistor and the LED. NOTE: the LED is asymmetric and must be inserted into the breadboard so that the leg under the "flat" edge is connected to the 820 ohm resistor and the round side is inserted into the +9V rail.
    • The second connection is to a wire that runs to a variable resistor (see the "finished circuit"). You will change the current into the OpAmp by varying the resistance through the circuit this point. You will start by putting no resistor in, where air serves as an infinitely large resistor.
  10. Double check your connections with the system diagram above before you power it up.
  11. To connect the 9V battery to the terminals, place the snap battery cover on the battery. Attach the alligator clip on the red battery lead to the red terminal of the breadboard, and the alligator clip on the black battery lead to the black terminal.

Examining the system behavior

Resistance = infinite Ω

Air connects the OpAmp's pin 6 to pin 2

  1. What happens to the LED when you power up the circuit?
  2. What happens to the LED when you shine the flashlight on the photodiode?
  3. Can you shine the flashlight on the photodiode so that the LED holds steady at 1/2 its maximal light intensity? The range of flashlight intensities that can hold the LED 1/2 lit is a measure of the "gain" in the system--where a narrow range of fully on to fully off is "digital" (switch-like) behavior, while a wide range of flashlight intensities that hold the LED 1/2 on is more "analog" (dial-like) behavior.
  4. Sketch a graph that has flashlight intensity on the x-axis and LED light intensity on the y-axis. At infinite resistance in place, is the circuit's behavior better described as a switch or a dial?
Resistance = 0Ω

Connect the OpAmp's pin 6 to pin 2 with a wire.

  1. What happens to the LED when you power up the circuit?
  2. What happens to the LED when you shine the flashlight on the photodiode?
  3. Can you shine the flashlight on the photodiode so that the LED holds steady at 1/2 its maximal light intensity?
  4. Add a line to your graph that has flashlight intensity on the x-axis and LED light intensity on the y-axis. With zero resistance in place, is the circuit's behavior better described as a switch or a dial?
Vary resistance

Connect the OpAmp's pin 6 to pin 2 with a 10 MΩ resistor.

  1. What happens to the LED when you power up the circuit?
  2. What happens to the LED when you shine the flashlight on the photodiode?
  3. Can you shine the flashlight on the photodiode so that the LED holds steady at 1/2 its maximal light intensity?
  4. Add one last line to your graph that has flashlight intensity on the x-axis and LED light intensity on the y-axis. Is this circuit's behavior better described as a switch or a dial?
  5. In natural language, can you describe how the effect of resistance on the gain is similar to the genetic experiment you are carrying out in this module?

DONE!

For Next Time

1. Draft your Materials and Methods section for your Mod 2 research article. You can describe the strains and plasmids you've used, the mechanics of taking a bacterial photograph, the b-gal assay and the library screen as far as through the electroporation. The due date for this assignment will vary depending on if you are presenting a Journal Club article next time as well.

  • If you ARE NOT giving a journal club talk next time, then this draft is due next time
  • If you ARE giving a journal club talk next time, then this draft is due before lab, one week from today.

2. If you are giving a journal club talk, the slides for your presentation should be uploaded to the Stellar website that is associated with our class. The presentation order will be determined by the order that your finished slides are uploaded.
3. (OPTIONAL): Comparisons of biological engineering and electrical engineering. Today's lab work may have made clear how much easier it is to assemble electrical circuits and run computer simulations of them than it is to assemble and test biological circuits. For example, it takes seconds to swap in a new resistor into your circuit or modulate an experimental parameter in the CAD tool, but it takes a few days to assemble DNA fragments (BioBricks or not). What other comparisons can you make that emphasize the

  • sensitivity of system performance
  • resolution
  • fabrication
  • repair
  • ???

of the two types of circuits.

Reagents

  • for electroporation
    • Strain NB188 (genotype: MC4100 ara+ Φ(OmpC-lacZ) 10-25 ΔenvZ::KanR +pPL-PCBamp)
    • pCph8 library: see Day 5
    • SOC media
      • 0.5% Yeast Extract
      • 2% Tryptone
      • 10 mM NaCl
      • 2.5 mM KCl
      • 10 mM MgCl2
      • 10 mM MgSO4
      • 20 mM Glucose
    • Tetrazolium(lac) + Amp + Cam plates (per liter)
      • 25.5 g Antibiotic Medium #2
      • 50 mg Tetrazolium indicator/L
      • 1 % lactose
      • 200 ug/ul ampicillin
      • 34 ug/ul chloramphenicol
Part Source Catalog # ~Cost
Breadboard Global (UDoIt) PB83E 17
Alligator clips Mueller Electric Co (DigiKey) 314-1010-ND 0.5
9V snap battery cover Philmore (UDoIt) BC120 1.5
9V battery Energizer (UDoIt) EN22, industrial 1.6
Flashlight LED keychain lights (Walgreen's) 693315-805697 2
LED AllElectronics LED-2, 5mm green T1 3/4 0.5
Resistor, 820 ohm NTE (UDoIt) QW182, 820 ohm, 2% 0.5
OpAmp Analog Devices Inc (DigiKey) AD8031ANZ 3
Resistor, 10 Mohm NTE (UDoIt) 2W610 1
Photodiode Leditech (AllElectronics) LT959X-91-0125 0.5
Wires Global (UDoIt) WK-3 3
Resistor color code card Elenco (UDoIt) CC-100 1
Clear case to hold parts Flambeau (UDoIt) DB221 2
Personal tools